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Introduction: Bacterial outer membrane vesicles (OMVs) are emerging as important players in the host-microbiome interaction, while also proving to be a promising platform for vaccine development and targeted drug delivery. The available methods for measuring their biodistribution, however, are limited. We aimed to establish a high-efficiency radiolabeling method for the treatment of OMVs. Methods: 99m Tc-HYNIC-duramycin was incubated with OMVs isolated from E. coli BL21(DE3) ΔnlpI ΔlpxM. Radiolabeling efficiency (RLE) and radiochemical purity (RCP) were measured with size-exclusion high-performance liquid chromatography. The biodistribution was quantitatively measured in mice using SPECT/CT imaging. Results: RLE was 81.84 ± 2.03% for undiluted OMV suspension and 56.17 ± 2.29% for 100× dilution. Postlabeling purification with a spin-desalting column results in 100% radioactivity in the OMV fraction according to HPLC, indicating 100% RCP of the final product. The biodistribution was found to be in line with previous data reported in the literature using other OMV tracking attempts. Conclusions: Our findings illustrate that using HYNIC-duramycin for labeling of the OMVs enhances efficiency and is easily implementable for in vivo imaging studies, significantly improving upon earlier methods., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in DNA repair. Here, we show that purified hSSB1 undergoes redox-dependent liquid-liquid phase separation (LLPS) in the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions in response to treatment modeling cellular oxidative stress. hSSB1's intrinsically disordered region is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we detected cytoplasmic hSSB1 condensates in various cell lines colocalizing with stress granules upon oxidative stress, implying extranuclear function in cellular stress response. Our results suggest condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Background: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs)., Methods: Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone)., Results: Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment., Conclusions: Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral β 3 -peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules., (© 2024. The Author(s).)

Pharmacologically active salicylanilides (2-hydroxy- N -phenylbenzamides) have been a promising area of interest in medicinal chemistry-related research for quite some time. This group of compounds has shown a wide spectrum of biological activities, including but not limited to anticancer effects. In this study, substituted salicylanilides were chosen to evaluate the in vitro activity on U87 human glioblastoma (GBM) cells. The parent salicylanilide, salicylanilide 5-chloropyrazinoates, a 4-aminosalicylic acid derivative, and the new salicylanilide 4-formylbenzoates were chemically and in vitro characterized. To enhance the internalization of the compounds, they were conjugated to delivery peptides with the formation of oxime bonds. Oligotuftsins ([TKPKG] n , n = 1-4), the ligands of neuropilin receptors, were used as GBM-targeting carrier peptides. The in vitro cellular uptake, intracellular localization, and penetration ability on tissue-mimicking models of the fluorescent peptide derivatives were determined. The compounds and their peptide conjugates significantly decreased the viability of U87 glioma cells. Salicylanilide compound-induced GBM cell death was associated with activation of autophagy, as characterized by immunodetection of autophagy-related processing of light chain 3 protein., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Schizophrenia (SCZ) is a severe neuropsychiatric disorder of complex, poorly understood etiology, associated with both genetic and environmental factors. De novo mutations (DNMs) represent a new source of genetic variation in SCZ, however, in most cases their biological significance remains unclear. We sought to investigate molecular disease pathways connected to DNMs in SCZ by combining human induced pluripotent stem cell (hiPSC) based disease modeling and CRISPR-based genome editing., Methods: We selected a SCZ case-parent trio with the case individual carrying a potentially disease causing 1495C > T nonsense DNM in the zinc finger MYND domain-containing protein 11 (ZMYND11), a gene implicated in biological processes relevant for SCZ. In the patient-derived hiPSC line the mutation was corrected using CRISPR, while monoallelic or biallelic frameshift mutations were introduced into a control hiPSC line. Isogenic cell lines were differentiated into hippocampal neuronal progenitor cells (NPCs) and functionally active dentate gyrus granule cells (DGGCs). Immunofluorescence microscopy and RNA sequencing were used to test for morphological and transcriptomic differences at NPC and DGCC stages. Functionality of neurons was investigated using calcium-imaging and multi-electrode array measurements., Results: Morphology in the mutant hippocampal NPCs and neurons was preserved, however, we detected significant transcriptomic and functional alterations. RNA sequencing showed massive upregulation of neuronal differentiation genes, and downregulation of cell adhesion genes. Decreased reactivity to glutamate was demonstrated by calcium-imaging., Conclusions: Our findings lend support to the involvement of glutamatergic dysregulation in the pathogenesis of SCZ. This approach represents a powerful model system for precision psychiatry and pharmacological research., Competing Interests: Declaration of competing interest The authors have no competing financial interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99m Tc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99m Tc-labeled-Duramycin in platelet imaging.

Hypothesis: In the last 20 years, it has been demonstrated that oppositely charged polyelectrolyte-surfactant (PE-S) mixtures are prone to forming kinetically arrested non-equilibrium aggregates, which are present in the prepared mixtures from rather low surfactant-to-polymer-repeat-unit ratios. Practically, this means that the PE-S mixtures used for the structural investigations of the formed PE-S complexes are typically a mixture of the primary PE-S complexes and large non-equilibrium aggregates of close to charge-neutral complexes., Experiments: In this work, we present a unique approach that allows the preparation of PE-S mixtures in the equilibrium one-phase region (surfactant binding β, is typically below 80%) without forming non-equilibrium aggregates. We used this method to prepare equilibrium, non-aggregated complexes of sodium poly(styrene sulfonate) (NaPSS, M w  = 17 kDa) and dodecyltrimethylammonium bromide (DTAB) (β = 10 - 70%) both in water and in an inert electrolyte (100 mM NaCl). The evolution of the complex structure was monitored by small-angle X-ray scattering (SAXS) as a function of increasing surfactant binding (β), and the measured scattering data were fitted by suitable structural models on an absolute scale where concentrations, compositions, and scattering contrasts calculated from molecular properties are used as restraints., Findings: We could show that at low binding (β < 30%), the system is a mixture of bare polyelectrolyte coils and NaPSS-DTAB complexes containing a closed surfactant associates of low aggregation number wrapped by the polyelectrolyte chain. Once all polymer chains are occupied by a micelle-like surfactant aggregate, the aggregation number increases linearly with increasing surfactant chemical potential. Using the structural insight provided by the SAXS measurements, we could fit the experimental binding isotherm data with a physically coherent, simple thermodynamic model. Finally, we also compared the stoichiometric NaPSS-DTAB precipitate's structure with the equilibrium complexes' structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64 Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64 Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs., (© 2023. The Author(s).)

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Extracellular vesicles (EVs), through their cargo, are important mediators of bystander responses in the irradiated bone marrow (BM). MiRNAs carried by EVs can potentially alter cellular pathways in EV-recipient cells by regulating their protein content. Using the CBA/Ca mouse model, we characterised the miRNA content of BM-derived EVs from mice irradiated with 0.1 Gy or 3 Gy using an nCounter analysis system. We also analysed proteomic changes in BM cells either directly irradiated or treated with EVs derived from the BM of irradiated mice. Our aim was to identify key cellular processes in the EV-acceptor cells regulated by miRNAs. The irradiation of BM cells with 0.1 Gy led to protein alterations involved in oxidative stress and immune and inflammatory processes. Oxidative stress-related pathways were also present in BM cells treated with EVs isolated from 0.1 Gy-irradiated mice, indicating the propagation of oxidative stress in a bystander manner. The irradiation of BM cells with 3 Gy led to protein pathway alterations involved in the DNA damage response, metabolism, cell death and immune and inflammatory processes. The majority of these pathways were also altered in BM cells treated with EVs from mice irradiated with 3 Gy. Certain pathways (cell cycle, acute and chronic myeloid leukaemia) regulated by miRNAs differentially expressed in EVs isolated from mice irradiated with 3 Gy overlapped with protein pathway alterations in BM cells treated with 3 Gy EVs. Six miRNAs were involved in these common pathways interacting with 11 proteins, suggesting the involvement of miRNAs in the EV-mediated bystander processes. In conclusion, we characterised proteomic changes in directly irradiated and EV-treated BM cells, identified processes transmitted in a bystander manner and suggested miRNA and protein candidates potentially involved in the regulation of these bystander processes.

Autophagy (cellular self-degradation) plays a major role in maintaining the functional integrity (homeostasis) of essentially all eukaryotic cells. During the process, superfluous and damaged cellular constituents are delivered into the lysosomal compartment for enzymatic degradation. In humans, age-related defects in autophagy have been linked to the incidence of various age-associated degenerative pathologies (e.g., cancer, neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immune deficiency) and accelerated ageing. Muscle mass decreases at detectable levels already in middle-aged patients, and this change can increase up to 30-50% at age 80. AUTEN-67 and -99, two small-molecule enhancers of autophagy with cytoprotective and anti-ageing effects have been previously identified and initially characterized. These compounds can increase the life span in wild-type and neurodegenerative model strains of the fruit fly Drosophila melanogaster . Adult flies were treated with these AUTEN molecules via feeding. Fluorescence and electron microscopy and Western blotting were used to assess the level of autophagy and cellular senescence. Flying tests were used to measure the locomotor ability of the treated animals at different ages. In the current study, the effects of AUTEN-67 and -99 were observed on striated muscle cells using the Drosophila indirect flight muscle (IFM) as a model. The two molecules were capable of inducing autophagy in IFM cells, thereby lowering the accumulation of protein aggregates and damaged mitochondria, both characterizing muscle ageing. Furthermore, the two molecules significantly improved the flying ability of treated animals. AUTEN-67 and -99 decrease the rate at which striated muscle cells age. These results may have a significant medical relevance that could be further examined in mammalian models.

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.

The aim of this study was to develop and characterize a Prussian Blue based biocompatible and chemically stable T1 magnetic resonance imaging (MRI) contrast agent with near infrared (NIR) optical contrast for preclinical application. The physical properties of the Prussian blue nanoparticles (PBNPs) (iron (II); iron (III);octadecacyanide) were characterized with dynamic light scattering (DLS), zeta potential measurement, atomic force microscopy (AFM), and transmission electron microscopy (TEM). In vitro contrast enhancement properties of PBNPs were determined by MRI. In vivo T1-weighted contrast of the prepared PBNPs was investigated by MRI and optical imaging modality after intravenous administration into NMRI-Foxn1 nu/nu mice. The biodistribution studies showed the presence of PBNPs predominantly in the cardiovascular system. Briefly, in this paper we show a novel approach for the synthesis of PBNPs with enhanced iron content for T1 MRI contrast. This newly synthetized PBNP platform could lead to a new diagnostic agent, replacing the currently used Gadolinium based substances., Competing Interests: The authors have declared that no competing interests exist.

RecQ helicases-also known as the "guardians of the genome"-play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to affect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility., (© 2022. The Author(s).)

Surface modification of silica nanoparticles with organic functional groups while maintaining colloidal stability remains a synthetic challenge. This work aimed to prepare highly dispersed porous hollow organosilica particles (pHOPs) with amino surface modification. The amino-surface modification of pHOPs was carried out with 3-aminopropyl(diethoxy)methylsilane (APDEMS) under various reaction parameters, and the optimal pHOP-NH 2 sample was selected and labelled with fluorescein isothiocyanate (FITC) to achieve fluorescent pHOPs (F-HOPs). The prepared pHOPs were thoroughly characterized by transmission electron microscopy, dynamic light scattering, FT-IR, UV-Vis and fluorescence spectroscopies, and microfluidic resistive pulse sensing. The optimal amino surface modification of pHOPs with APDEMS was at pH 10.2, at 60 °C temperature with 10 min reaction time. The positive Zeta potential of pHOP-NH 2 in an acidic environment and the appearance of vibrations characteristic to the surface amino groups on the FT-IR spectra prove the successful surface modification. A red-shift in the absorbance spectrum and the appearance of bands characteristic to secondary amines in the FTIR spectrum of F-HOP confirmed the covalent attachment of FITC to pHOP-NH 2 . This study provides a step-by-step synthetic optimization and characterization of fluorescently labelled organosilica particles to enhance their optical properties and extend their applications.

Porous hollow silica particles possess promising applications in many fields, ranging from drug delivery to catalysis. From the synthesis perspective, the most challenging parameters are the monodispersity of the size distribution and the thickness and porosity of the shell of the particles. This paper demonstrates a facile two-pot approach to prepare monodisperse porous-hollow silica particles with uniform spherical shape and well-tuned shell thickness. In this method, a series of porous-hollow inorganic and organic-inorganic core-shell silica particles were synthesized via hydrolysis and condensation of 1,2-bis(triethoxysilyl) ethane (BTEE) and tetraethyl orthosilicate (TEOS) in the presence of hexadecyltrimethylammonium bromide (CTAB) as a structure-directing agent on solid silica spheres as core templates. Finally, the core templates were removed via hydrothermal treatment under alkaline conditions. Transmission electron microscopy (TEM) was used to characterize the particles' morphology and size distribution, while the changes in the chemical composition during synthesis were followed by Fourier-transform infrared spectroscopy. Single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied to assess the monodispersity of the hollow particles prepared with different reaction parameters. We found that the presence of BTEE is key to obtaining a well-defined shell structure, and the increase in the concentration of the precursor and the surfactant increases the thickness of the shell. TEM and spICP-MS measurements revealed that fused particles are also formed under suboptimal reaction parameters, causing the broadening of the size distribution, which can be preceded by using appropriate concentrations of BTEE, CTAB, and ammonia.

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects.

We demonstrated that the plasma membrane Ca 2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca 2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca 2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

Here we describe the generation of induced pluripotent stem cell (iPSC) lines from peripheral blood samples of identical twin sisters with type 2 diabetes mellitus (DM2). Two clonal lines from each patient (HU-DM2-A-1, HU-DM2-A-2 and HU-DM2-B-1, HU-DM2-B-2) were established via Sendai viral reprograming of peripheral blood mononuclear cells, and characterized to confirm pluripotency and genetic integrity. The established iPSC lines can help to investigate DM2 related cellular phenotypes and provide a model system for drug testing., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors., Methods: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca 2+ ) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines., Results: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca 2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology., Conclusions: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family., Limitations: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.

Objectives: Ionising radiation-induced alterations affecting intercellular communication in the bone marrow (BM) contribute to the development of haematological pathologies. Extracellular vesicles (EVs), which are membrane-coated particles released by cells, have important roles in intercellular signalling in the BM. Our objective was to investigate the effects of ionising radiation on the phenotype of BM-derived EVs of total-body irradiated mice., Methods: CBA mice were irradiated with 0.1 Gy or 3 Gy X-rays. BM was isolated from the femur and tibia 24 h after irradiation. EVs were isolated from the BM supernatant. The phenotype of BM cells and EVs was analysed by flow cytometry., Results: The mean size of BM-derived EVs was below 300 nm and was not altered by ionising radiation. Their phenotype was very heterogeneous with EVs carrying either CD29 or CD44 integrins representing the major fraction. High-dose ionising radiation induced a strong rearrangement in the pool of BM-derived EVs which were markedly different from BM cell pool changes. The proportion of CD29 and CD44 integrin-harbouring EVs significantly decreased and the relative proportion of EVs with haematopoietic stem cell or lymphoid progenitor markers increased. Low-dose irradiation had limited effect on EV secretion., Conclusions: Ionising radiation induced selective changes in the secretion of EVs by the different BM cell subpopulations., Advances in Knowledge: The novelty of the paper consists of performing a detailed phenotyping of BM-derived EVs after in vivo irradiation of mice.

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)